Translating information from two-dimensional (2D) culture into three-dimensional (3D) systems has been a major hurdle in the use of biopolymers for tissue repair applications. In order to design improved culture environments and responsive architectures for neuronal repair, our goal is to advance the understanding of how neurons respond to 3D environments. We hypothesize that 3D culture 1) imposes changes in matrix ligand organization that directly alter neuronal behavior by modulating 1 integrin-cytoskeletal signaling and 2) imposes changes in dissolved oxygen profiles. Therefore substrate dimensionality is a critical factor for neuronal survival and re-establishment of functional connectivity required for the success of cell-based neural therapies. To test this hypothesis, we will first investigate the roles of 1 integrin, vinculin, FAK and pFAK in DRG neurite outgrowth in 3D laminin culture scaffolds (Aim 1). We will then optimize the 3D culture scaffolds to maximize neurite outgrowth and determine whether the type of 1 integrin ligands impacts integrin signaling during neurite outgrowth in 3D scaffolds (Aim 2). Finally, we will determine how oxygen concentration impacts neuronal survival and outgrowth in 3D culture by applying novel oxygen-sensing microparticles to directly measure spatial and temporal dissolved oxygen profiles (Aim 3). Our preliminary studies indicate that 3D culture imposes changes in 1 integrin signaling that result in altered neurite outgrowth. To study this effect in more detail, we have established two novel tools to provide quantitative data in a physiologically relevant 3D system. First, we have developed a 3D culture system with controllable physical and biochemical material properties. Second, we have developed novel fluorescent oxygen-sensing microparticles to detect spatial and temporal changes in dissolved oxygen content. The microparticles demonstrate sensing performance comparable to traditional electrochemical probes, but are biocompatible and allow rapid, automated and non-invasive measurements local to cells and without consuming oxygen. Based on these studies, we will use cellular and environmental markers of neural morphology and dissolved oxygen to design a system that recapitulates tissue physiology. Our studies will delineate key signaling mechanisms to provide a biological basis for testing new 3D nerve repair therapies. Moreover, the adaptability of the proposed tunable synthetic gels allows for the addition of other biomolecules, pharmaceuticals, reporter constructs and cell types. Thus, the tunable synthetic gels will have broad utility towards investigations of permissive/inhibitory matrix cues as well as neuronal-glial interactions in normal and diseased states. The proposed project will provide new fundamental knowledge about neuronal response to 3D microenvironments and will enable the improved design of future biomaterials-based approaches for neural repair. PUBLIC HEALTH RELEVANCE: Much of our current understanding of neurobiology relies on disrupted tissues, laboratory studies in artificial environments, and clinical observations. We hypothesize that the next generation of nerve repair therapies relies on the design of materials that better replicate the three-dimensional structure and physiology of native tissues. The goals of this proposed work is to advance the understanding of neuronal response to three-dimensional environments and to provide new improved materials and tools to study and repair neurons.